ANTIBACTERIAL COMPOSITION CONTAINING MULTIMERIC ALPHA-LACTALBUMIN

Abstract: The present invention relates to the use of a multimeric alpha-lactalbumin in the preparation of preparations to be used in therapeutic or prophylactic treatment and/or for diagnostic use for infections, preferably of the respiratory tract, caused by bacteria, in particular S. pneumoniae and/or H. influenzae.

Description: Antibacterial composition containing multimeric alpha-lactalbumin. DESCRIPTION Technical field The present invention relates to a novel antibacterial protein and compositions, in the form of pharmaceutical compositions, human food compositions, and animal feedstuffs comprising said protein to be used in the therapeutic and/or prophylactic treatment of infections caused by bacteria, in particular Streptococcus pneumoniae and/or Haemophilus influenzae, as well as a method for diagnosing infections caused by said bacteria. The object of the present invention is to obtain a protein and compositions containing said .protein for prophylactic and/or therapeutic treatment of infections caused by bacteria, in par- ticular Streptococcus pneumoniae and Haemophilus influenzae in the upper airways, ear-nose-and-throat infections, but also in the lower airways, e.g., the lungs by preventing adhesion of and/or causing a bactericidal effect on these bacteria. A fur¬ ther object is to be able to diagnose infections caused by these bacteria. Background of the invention Natural antimicrobial compounds exist in secreted form as well as in cells of immune and non-immune origin. Human milk has been used as a source for the purification of such compounds. These previously known compounds include speci¬ fic antibodies to the micro-organism surface structure, casein, lysozyme, and oligosaccharides. The mechanism of action differs between the groups of antimicrobial molecules. Antibodies and receptor analogues prevent micro-organism adherence to mucosal surfaces. Lysozyme attacks the cell wall etc. The term bacterial adherence denotes the binding of bacteria to mucosal surfaces. This mechanic association is a means for the organism to resist elimination by the body fluids, and to es¬ tablish a population at the site where relevant receptors are expressed. In most cases where the mechanisms of attachment have been identified it is a specific process. The bacterial ligands , commonly called adhesins bind to host receptors. For Gram-negative bacteria, the adhesins are commonly associated with pili or fimbriae, rigid surface organelles that help bac¬ teria to reach the appropriate receptor in the complex cell surface. The fimbriae function as lectins, i.e. they show spe¬ cificity for receptor epitopes provided by the oligosaccharide sequences in host glyco-conjugates (13). For Gram-positive bac- teria, on the other hand, the adhesins are not expressed as a surface organell, but rather linked to cell wall components and lipoteichoic acids (21,22). The receptor epitopes for Gram pos¬ itive bacteria may consist of oligosaccharide sequences but can also be provided by peptides e.g. in connective tissue proteins (10). The functional consequences of adherence depend on the virulen¬ ce of the bacterial strain, and on the form of the receptor. When cell-associated, the ligand receptor interaction facili- tates colonization and tissue attack (8). When secreted the re¬ ceptor molecule will occupy the adhesins, and competitively in¬ hibit attachment to the corresponding cell-bound receptor. Hu¬ man milk is a rich source of such competing soluble receptor molecules. The ability of specific antibodies to inhibit attachment is well established. This was first demonstrated for Vibrio chole- ra and oral streptococci. The anti-adhesive antibodies may act in either of two ways: 1) Antibodies to the receptor binding sites of the adhesin com¬ petitively inhibit receptor interaction or 2) antibodies to bacterial surface molecules which are not di¬ rectly involved in adherence may agglutinate the bacteria and thereby reduce the number of organisms available for binding. In either of the above cases the anti-adhesive activity of the antibody is attributed to the specificity of the antigen-com- bining site. Recently an alternative mechanism of interaction between secretory IgA and E_ ; _ coli based on lectin-carbohydrate interactions was identified. Human milk drastically inhibits the attachment of Streptococcus pneumoniae and Haemophilus influenzae to human nasopharyngeal epithelial cells. It contains antibodies to numerous surface antigens on these organisms, e.g., the phosphoryl choline and capsular polysaccharides of S^ pneumoniae, the lipopolysaccha- ride and outer membrane proteins of H^ influenzae. Accordingly, some of the anti-adhesive activity in milk resides in the immunoglobulin fraction. The remaining anti-adhesive activity in the non-immunoglobulin fraction of milk may be explained by two types of molecules: free oligosaccharides and glycoproteins in the casein fraction. Human milk is unique with regard to its content of complex car¬ bohydrates. The free oligosaccharide fraction of milk is do i- nated by the lactoseries and with improving methods of isola¬ tion and characterization of carbohydrates more than 130 oligo¬ saccharides containing up to 20 monosaccharides per molecule have been identified. An• anti-adhesive activity against S^ pneumoniae in a low mole¬ cular weight fraction (<5 kDa ) of milk was explained by the free oligosaccharides. In contrast there was no such effect against H^ influenzae (15). An anti-adhesive activity of high molecular weight components of milk was localized to the casein fraction. Human casein drastically reduced the adherence both of S^ pneumoniae and H. influenzae (15). This effect was species specific. Alpha-lactalbumin is a mettaloprotein , which shows some degree of heterogeneity depending on Ca(II) saturation and/or glyco- sylation (1). Alpha-lactalbumin acts as a specifier protein in the lactose synthase system. During lactation, alpha-lactalbu¬ min is formed in the mammary gland and it alters the substrate specificity of the galactosyltransferase enzyme from N-acetyl glucosamine (GlcNAc) to glucose (Glc), enabling lactose syn- thesis to take place: GT UDP-Gal + Glc > Lactose + UDP alphaLA Multiple forms of bovine, pig, sheep and goat alpha-lactalbumin have been isolated and well characterized (2, 3). These multip¬ le forms differ in a few amiho residues or the number of di- sulphide bonds (4, 5) but are all active in the lactose syn- thase system. The physiological relevance or functions of these different forms of alpha-lactalbumin are not known. Alpha-lact¬ albumin has undergone a high rate of evulotionary change and it shows homology with lysozyme (1). These two proteins are thought to originate from the same ancestral protein. Whereas lysozyme is known as an anti-bacterial agent, alpha-lactalbumin has not yet been found to have anti-bacterial functions. Description of the present invention The present invention describes the identification of a new anti-bacterial protein or group of proteins from milk. The pro¬ tein comprises a multimeric form of alpha-lactalbumin. In the following this protein, or group of proteins, is ab¬ breviated ALLP, Anti-adhesive Lactalbumin Like Protein. The term antimicrobial or anti-bacterial protein used in the context of the present invention means here and in the follow¬ ing a protein which inhibits adherence of micro-organisms to tissue and/or exerts a bactericidal effect on microorganisms. Further characteristics of the invention will be evident from the accompanying claims. The present invention will be described more in detail with re¬ ference to the example given below. EXPERIMENTAL Purification of the active anti-adhesive and bactericidal pro¬ tein (ALLP) Milk samples from lactating women were screened for anti-ad¬ hesive activity against S^ pneumoniae and H_ ; _ influenzae. About 50 1 of breast milk with high anti-adhesive activity was col- lected from one healthy donor and used for the purification of ALLP. About 5 1 of milk was thawed at a time and centrifuged to remove fat. Casein was prepared from the defatted milk by acid precipitation at pH 4.6. ALLP was purified as outlined below: (i) Ion-exchange chro atography of casein. Casein was fractionated using an ion-exchange column (14 cm x 1.5 cm) packed with DEAE-Tris-acryl M (LKB, Sweden) attached to an FPLC (Pharmacia, Sweden) using a NaCl gradient". 100 mg of the lyophilized casein was dissolved in 10 ml of 0.01 M Tris- HCl, pH 8.5. After centrifugation, the sample was directly app¬ lied to the column and the run was under the following condi¬ tions: buffer A: 0.01 M Tris-HCl, pH 8.5; Buffer B: buffer A containing 1 M NaCl/1. Gradient program: from 0-3 ml 100%A, from 3-60 ml 15% B; from 60-85 ml 25% B; from 85-87 ml 100% B; from 87-89 ml 100% B for 2 min; from 89-120 ml 100% A. The gra¬ dient was not linear, but was interrupted at the elution of each peak for better separation. Flow rate: 1 ml/min, recorder 0.2 cm/min. The buffers were degassed and filtered through a 0.22.urn filter before use. The peaks were monitored at 280 nm and the fraction size was 3 ml. Fractions were pooled as shown (FIG. 1A). The pools (I-VI) were then desalted by dialysis (membrane cut off 3.5 kD) against distilled water for at least 48 hrs, lyophilized and tested for anti-adhesive activity. ( ii ) Gel chromatography of pool VI 100 mg of the active pool VI obtained after repeated FPLC frac- tionations of casein, were dissolved in 7 ml 0.06 M sodium phosphate buffer, pH 7.0 and applied to a Sephadex R G-50 (Phar¬ macia, Sweden) column (93 cm x 2.5 cm). Flow rate was 30 ml/hr, peaks were monitored at 280 nm, 3 ml fractions were collected and pooled as shown (FIG. 2A). The pools were desalted by dia- lysis, lyophilized, tested for composition and for anti-adhe¬ sive activity. Ion-exchange chromatography of commercial alpha-lactalbumin. 20 mg of commercial (Sigma) human or bovine alpha-lactalbumin were dissolved in 2 ml 0.01 M Tris-HCl, pH 8.5. The ion-ex¬ change chromatography of alpha-lactalbumin was under similar conditions as described above for the fractionation of casein. The NaCl gradient was linear ;(not interrupted ) , flow rate was 1 ml/min, 3 ml fractions were collected and pooled as shown in FIG. IB. The pools were dialysed, (membrane cut-off 3.5 kD), lyophilized, resuspended to the required concentration and tested for anti-adhesive activity. Gel chromatography of commercial alpha-lactalbumin Approximately 8-10 mg of commercial human or bovine alpha- lactalbumin (Sigma) were dissolved in 3 ml 0.06 M sodium phos- phate buffer, pH 7.0 and fractionated on the Sephadex G-50 column as described above. Flow rate was 30 ml/hr, peaks were monitored at 280 nm, 3 ml fractions were collected and pooled as. shown (FIG. 2B) . The pools were desalted by dialysis (mem¬ brane cut-off 3.5 kD) against distilled water for at least 48 hrs , lyophilized, tested for composition and for anti-adhesive activity. 6-8 mg retained of the material retained and eluting after 1 M NaCl during ion-exchange chromatography of alpha- lactalbumin were dissolved in 5 ml 0.06 M sodium phosphate buffer pH 7.0 and subjected to gel chromatography on the G-50 column as described above. 3 ml fractions were collected and pooled (FIG. 3). The pools were desalted, lyophilized, and tested for anti-adhesive activity. Polyacrylamide gradient gel electrophoresis (PAGGE). Analytical PAGGE was performed using 4-20% polyacrylamide pre- cast gels (Bio-Rad, Richmond, CA) on a Bio-Rad Mini Protean II cell. To 10 .ul (5-10 mg/ml) each of the lyophilized fractions, an equal volume of sample buffer (13.1% 0.5 M Tris-HCl, pH 6.8, 10.5% glycerol, 1.2% SDS and 0.05% bromophenol blue) was added. 20 .ul of each was then loaded on to the gel which was run in Tris-glycine buffer (pH 8.3) with 0.1%SDS at 200V constant vol¬ tage for about 40 min. Staining of the proteins was made by im¬ mersing the gel in Coomassie Blue solution (0.1% in 40% methan- ol, 10% acetic acid) for about 0.5 hr. Destaining was by seve- ral changes in 40% methanol, 10% acetic acid until a clear background was obtained. Ion desorption mass spectrometry ALLP and commercial alpha-lactalbumin were analyzed by ion- desorption mass spectrometry. Bacteria S. pneumoniae (CCUG3114 and 10175) and H influenzae (Hil98) we¬ re used throughout the experiments. These strains were known to adhere well to human nasopharyngal epithelial cells ixi vitro. These strains were initially isolated from the nasopharynx of children with frequent episodes of acute otitis media. The strains were kept lyophilized and were transferred to blood agar (10175) or Levinthal medium agar plates (Hi 198). S^ pneu- moniae was cultured for 9 hrs at 37 C in liquid medium (17), harvested by centrifugation and suspended in 1 ml of 0.9% NaCl with 1% choline. H_^ influenzae Hil98 was cultured for 4 hrs in haemophilus medium (18), harvested by centrifugation and sus¬ pended in phosphate-buffer saline, (PBS). Adhesion inhibition Adhesion and inhibition of adhesion was tested as previously described (15, 19). In brief, epithelial cells from the oro- 5 pharynx of healthy donors (10 /ml) were mixed with the bacte- 9 rial suspensions (10 /ml). After incubation of bacteria and epithelial cells, unbound bacteria were eliminated by repeated cycles of centrifugation and resuspension in NaCl with 1% cho- line ( 10175 ) or PBS ( Hi 198 ) . The inhibitory activity of the different fractions was tested by preincubation with bacteria for 30 min at 37 C prior to ad- dition of epithelial cells. The number of epithelial cells at¬ tached was counted with the aid of an interference contrast mi¬ croscope (Ortolux II microscope with interference contrast equipment TE Leitz, Wetzlar). Adherence was given as the mean number of bacteria/cell for 40 epithelial cells. Inhibition was given in per cent of the value of the buffer control. RESULTS Properties of ALLP ALLP was purified from human milk by fractionation of casein by ion-exchange chromatography and fractionantion of the pool eluting after 1 M NaCl by gel chromatography. The ion-exchange fractionation profile of casein is shown in FIG 1A. Eluted fractions were pooled as indicated and tested for anti-adhesive activity. Pool VI retained the anti-adhesive activity of ca- sein; this pool inhibited the attachment of S^ pneumoniae and H. influenzae by more than 80% of the control (Table 3). The remaining fractions were inactive and were not analyzed fur¬ ther. Pool VI was fractionated by gel chromatography on the Sephadex G-50 column. The fractionation profile showed two distinct well separated peaks (FIG. 2A). Eluted fractions were pooled as shown, desalted, and tested for anti-adhesive activity. Pool K retained 98% ,ρf the anti-adhesive activity against S_^ pneumo- niae and 91% of the activity against H^ influenzae. Pool L was inactive (Table 3). Analytical PAGGE of pool K showed the presence of bands in the 14-15 kD region, one band in the 30 kD region, and two bands stained in the 100 kD region. Pool L showed the presence of only one band in the 14-15 kD region (FIG. 2A, inset). The N- terminal amino acid sequence analysis showed that the bands of pool K were similar and were identical to the N-terminal se¬ quence of human alpha-lactalbumin. The active anti-adhesive protein in pool K was designated as Anti-adhesive Lactalbumin Like Protein (ALLP). ALLP reduced attachment of both S^ pneumoniae and H^ influenzae by about 60% at a concentration of 1 mg/ml. Mass spectrometry of ALLP The results from analytical PAGGE suggested that ALLP might oc- cur in a multimeric form. By ion laser desorption mass spec¬ trometry, ALLP showed three distinct mass fragments (1, 2 and 3) at 14128.7 m/z, 28470.5 m/z and 42787.8 m/z, respectively (FIG. 4). These fragments agreed with the monomeric (14 m/Z), dimeric (28 m/z) and trimeric (42 m/z) mass ranges of the pro- tein. Comparison of ALLP and commercial alpha-lactalbumin When tested for anti-adhesive activity, commercial alpha-lact¬ albumin did not inhibit the adherence of S^ pneumoniae or H_ ; _ influencae even at a concentration of 10 mg/ml (Table 4). ALLP showed stained bands in the 14-15 kD, 30 kD and the 100 kD re¬ gions, whereas the commercial alpha-lactalbumin stained only one band in the 14-15 kD region. The N-terminal amino acid se¬ quence of ALLP showed complete homology with the sequence of human alpha-lactalbumin. The lack of anti-adhesive activity of commercial alpha-lact¬ albumin, as compared to ALLP, might be due to a difference in their molecular forms. Therefore commercial human alpha-lact- albumin was subjected to ion laser desorption mass spectro¬ metry. The spectrum showed only one mass fragment at 14128.7 m/z corresponding to the monomeric form of alpha-lactalbumin (calculated molecular mass = 14.079 kD). Thus commercial human alpha-lactalbumin was in the monomeric form and lacked anti- adhesive activity, whereas, ALLP was found to be multimeric and inhibited the attachment of S^ pneumoniae and H^ influenzae to human oropharyngeal cells _in_ vitro. Ion-exchange chromatography of human alpha-lactalbumin In order to test the effect of ion exchange chromatography on the anti-adhesive effect of commercial human alpha-lactalbumin, 20 mg of the commercial sample was applied onto the Tris-acryl column. The ion-exchange profile is shown in FIG. IB. About 50% of the material applied was retained on the column and eluted after the application of 1 M NaCl (arrow, FIG. IB). The diffe¬ rent fractions were pooled as shown. After desalting and lyo- philization the fractions were reconstituted to a concentration of about 5-10 mg/ml and tested for anti-adhesive activity. Anti-adhesive effect of human alpha-lactalbumin after ion-ex¬ change chromatography Before ion-exchange chromatography commercial human alpha-lact- albumin lacked anti-adhesive activity (Table 4). After it was subjected to ion-exchange chromatography, the pool which was retained and eluted with 1 M NaCl (pool LA_, FIG. IB) inhibited the attachment of both S^ pneumoniae and H^ influenzae by more than 95% of the value of the control (Table 4). The other pool (LA. ) obtained was inactive. Gel chromatography of human alpha-lactalbumin before and after ion-exchange chromatography Since about 50% of the commercial human alpha-lactalbumin had become active after ion-exchange chromatography it was decided to check the mobility of the alpha-lactalbumin and pool LA,, on gel chromatography. The G-50 gel chromatographic profile of human alpha-lactalbumin before ion-exchange chromatography is shown in FIG. 2B. The alpha-lactalbumin eluted as a single peak, which gave a single band (14-15 kD) on PAGGE analysis (inset, FIG. 2B) . This pool LA was found to be inactive when tested for anti-adhesive acti¬ vity (Table 4). The gel chromatographic profile of the active pool LA obtain¬ ed after ion-exchange chromatography of alpha-lactalbumin is shown in FIG. 3. This pool eluted as two well separated peaks (1 and 2, FIG. 3) corresponding to the eluting volumes of peaks K and L of the casein (FIG. 2A). When tested for anti-adhesive activity pool 1 retained the activity against both S_ ; _ pneumo- niae and H^ influenzae, whereas pool 2 was inactive (Table 4). When pool 1 was analysed by analytical PAGGE a pattern similar to that of ALLP was obtained, bands stained at 14-15 kD region, 30 kD region, and two bands at 100 kD region. Pool 2 gave a single band at the 14-15 kD region, corresponding to monomeric alpha-lactalbumin (inset, FIG. 3). Properties of commercial bovine alpha-lactalbumin. Since commercial human alpha-lactalbumin could be converted to the active multimeric form by ion-exchange chromatography it was decided to test the activity of bovine alpha-lactalbumin and to test its mobility on ion-exchange and gel chromatogra¬ phy. When tested for anti-adhesive activity, bovine alpha-lact¬ albumin was found to be inactive in inhibiting the attachment of S_ ; _ pneumoniae and H_ ; _ influenzae (Table 5). 20 mg of bovine alpha-lactalbumin were subjected to ion-ex¬ change chromatography under similar conditions described above for human alpha-lactalbumin. 50% of the material applied to the column was retained and eluted after 1 M NaCl. The elution pat¬ tern was similar to that obtained for human alpha-lactalbumin (FIG. IB). Pool BL_ of bovine alpha-lactalbumin, corresponding to the elution volume of pool LA_ of human alpha-lactalbumin (FIG. IB) inhibited the attachment of S^ pneumoniae by more than 95% and of H_ ; _ influenzae by more than 80% of the value of the control (Table 5). When subjected to gel chromatography on the G-50 column as de¬ scribed above, bovine alpha-lactalbumin eluted as a single peak corresponding to the elution volume of human alpha-lactalbumin (FIG. 2B) . In contrast, the material in pool BL resolved into two distinct peaks corresponding to pools 1 and 2 obtained for human alpha-lactalbumin (FIG. 3). The pool eluting just after the void volume of the column (corresponding to pool 1) retain¬ ed the anti-adhesive activity, whereas, the other pool was in¬ active. The active pool had a PAGGE pattern similar to that of ALLP, whereas, the inactive pool stained only one band in the 14-15 kD region. Thus a portion of the commercial bovine alpha-lactalbumin was also converted to the active multimeric form by ion-exchange chromatography. Bactericidal effect The present ALLP was tested with regard to bactericidal effect on different strains of S^ pneumoniae being known to be resist ant to antibiotics, and some other strains of Streptococcus, E coli, H. influenzae and M^ cat . Thereby the different bacterial strains were inoculated onto growth plates after incubation with ALLP in different concent- rations. The viable counts (CFU) were determined at inocula¬ tion, 0.5 h, 2 h, and 4 h (hours), respectively, after inocula tion. Table 1 below shows the viable counts after incubation t a medium containing 10 mg/ml of ALLP compared with the control Table 1 Viable counts (CFU) on S^ pneumoniae strains after exposure to ALLP. Strain Viable counts (CFU) designation Oh 0.5h 2h 4h 4 10175 control 2xl0 6 lxlO 6 lxlO 5 1x10 ALLP 2xl0 5 - - - 15006-92 control lxlO 4 2xl0 4 lxlO 3 - 4 ALLP 2x10 _ _. _ Table 1 cont'd Strain Viable counts (CFU) designation Oh 0.5h 2h 4h 14060-92 control 2x10 1x10 " 1x10 ALLP 2xl0 5 15256-92 control lxlO 6 2x10 2x10- 4x10 ALLP 2xl0 6 14326-92 control 4xl0 5 2x10 ' 2x10 2x10 " 4 ALLP 7x10 Prag 1828 control 5x10 2x10 5x10 " ALLP 5x10 14091-92 control 3xl0 : 5x10- 1x10 ' ALLP 7x10 " 14117-92 control 2x10 - 2x10 2x10 ALLP 2x10 14612-92 control 3x10 1x10 " 2x10 1x10 ' ALLP 3x10 Dk 84/87 control 1x10 5x10 2x10 6x10 ALLP 3x10 14007-92 control 1x10 5x10 4x10 " ALLP 1x10 14030-92 control 5x10 2x10 2x10 " ALLP 5x10 2xl0 ] 14423-92 control 6x10 6xl0 ( 1x10 6x10 " ALLP 2x10 3x10 " 4502- 93 control 4x10 ALLP 5x10 SA44165 control 2x10 5x10' ALLP 3x10 1017-92 control 1x10 5x10" 4x10 " ALLP 9x10 317-93 control 4x10 1x10 5x10 " ALLP 2x10 760-92 control 2x10 2x10 1x10 1x10 ALLP 8x10 Table 1 cont'd Strain Viable counts (CFU) designatioi 1 Oh 0.5h 2h 4h Hun 859 control 6xl0 5 3xl0 5 2xl0 5 2xl0 5 ALLP 3xl0 5 - - - Hun 963 control lxlO 7 4xl0 6 lxlO 5 - ALLP 5xl0 6 - - - 4 4 BN 241 control 4xl0 6 5x10 2x10 - ALLP 2xl0 5 - - - Table 2 Viable counts (CFU) on different bacterial species Strain Viable counts (CFU) designation Oh 0.5h 2h 4h S. mitis control lxlO 6 lOxlO 6 2xl0 5 lxlO 5 116 ALLP lxlO 6 - - - S. sanguis control 5xl0 7 3xl0 7 4xl0 7 5xl0 6 197 ALLP 3xl0 7 2xl0 5 2xl0 2 - E. coli control 6xl0 6 5xl0 6 3xl0 6 3xl0 6 60 ALLP 7xl0 6 5xl0 6 lxlO 7 2xl0 7 4 control 5xl0 6 5xl0 6 5xl0 6 7xl0 6 ALLP 5xl0 6 6xl0 6 lxlO 7 2xl0 7 H. ' influenzae control 4xl0 7 lxlO 7 4xl0 6 2xl0 5 21594 ALLP 3xl0 7 4xl0 5 <lxl0 3 <lxl0 3 21300 control 4xl0 7 2xl0 7 5xl0 6 3xl0 5 ALLP 4xl0 7 2xl0 6 2xl0 4 2xl0 3 4 M. cath. control 4xl0 5 3xl0 5 5xl0 4 2x10 71257 C+ ALLP 3xl0 5 2xl0 5 3xl0 3 - 71295 C+ control 2xl0 7 lxlO 7 3xl0 6 6xl0 5 ALLP 2xl0 7 5xl0 6 2xl0 6 3xl0 5 C+ = beta-lactamase producing A dose response curve was made up based on the bactericidal ef- feet on S^ pneumoniae 10175 at different levels of admini¬ stration of ALLP compared with control (no addition). Thereby ALLP was administered at 0.1 mg/ml, 0.5 mg/ml, and 1.0 mg/ml, respectively. The graph obtained is shown in FIG. 5. As evident therefrom as little as 0.1 mg/ml of ALLP provides a bacteri¬ cidal effect on S^ pneumoniae. The viable counts were further determined using different con¬ trol proteins, viz. bovine serum albumine (BSA), alphalactal- bu ine (bovine origin), lactoferrin (bovine origin) in a con¬ centration of 10 mg/ml, and control (no protein). As evident from FIG. 6 these proteins had no bactericidal effect on S. pneumoniae 10175. A new form of alpha-lactalbumin (ALLP) with anti-adhesive ac¬ tivity and bactericidal effect against the respiratory tract pathogens S^ pneumoniae and H^ influenzae was thus isolated and characterized from a human milk sample. Commercial human or bovine alpha-lactalbumin lacked anti-adhesive activity in the assay system. A portion of the commercial human and bovine alpha-lactalbumin was converted to active form by ion exchange chromatography. The active and non-active forms of alpha- lactalbumin showed different mobilities on gel chromatography and their staining patterns on gel electrophoresis were also different. By ion-desorption mass spectrometry analysis, ALLP was found to be in the trimeric form, whereas commercial alpha- lactalbumin was monomeric. The activated forms of commercial human and bovine alpha-lactalbumin showed gel pattern similar to the trimeric form. A portion of the monomeric form of alpha- lactalbumin was separated from the multimeric form and was found to be inactive in inhibiting the adherence of both S. pneumoniae and H. influenzae. The three forms of alpha-lact- albumin (mono, di and tri ) existed in some sort of equilibrium after ion-exchange chromatography and could not successfully be separated from each other. This proposes that the active anti- adhesive alpha-lactalbumin (ALLP) is a multimeric form not pre¬ viously identified in human milk. The identification of ALLP in a previous casein preparation was a result of its purification being monitored by the biological activity (16). It retained all of the anti-adhesive activity of casein and thus could be followed during the purification pro¬ cedures. This form of alpha-lactalbumin has not previously been disclosed to be present in human milk. The early studies of the present inventors showed that the anti-adhesive effect of human milk against S _ pneumoniae and H_ ; _ influenzae was independent from the specific antibody activity and was concentrated in a casein fraction (15). Casein was, however, found to have both a bactericidal effect and an anti-adhesive effect. A bactericidal effect was present and was found to be more pronounced against S. pneumoniae than H^ influenzae. The anti-adhesive activity remained intact after removal of the fatty acids from casein. The mechanism of adhesion inhibition of ALLP was found to be independent from its carbohydrate content. Carbohydrate ana- lysis of ALLP showed the presence of only one monosaccharide unit associated with the molecule. Removal of this monosaccha¬ ride unit by glucosidase treatment did not alter the anti-ad¬ hesive effect of ALLP. Also since the commercial forms of human and bovine alpha-lactalbumin could be activated by ion-exchange chromatography, it is very unlikely that the carbohydrate play any role in the anti-adhesive or bactericidal effect of ALLP tested by the biological analysis system. Being predominantly a whey protein, alpha-lactalbumin is usu- ally purified from the alpha-lactalbumin rich fractions of whey. Since the monomeric form and the multimeric forms have different mobilities on gel chromatography, the active multi¬ meric forms are lost during the purification procedures. It is thus not surprising that the commercial preparations of alpha- lactalbumin lacked anti-adhesive properties in the present sys¬ tem. Genetic variants of alpha-lactalbumin have been isolated from milk of other mammals including bovine. Most of these forms consist of four disulphide bonds and a form of bovine alpha-lactalbumin with three disulphide bonds have also been isolated (5). The physiological role of these different forms of alpha-lactalbumin is not known. The present data demonstrate that the monomeric alpha-lactalbumin completely lacked biologi- cal activity in the present system. Aggregation and polymeriza¬ tion may therefore be an important event in the anti-adhesive activity of ALLP against S^ pneumoniae and H^ influenzae. The present data demonstrate that the multimeric alpha-lact¬ albumin is active in adhesion inhibition of the respiratory tract pathogens and can thus play a role in the protection against respiratory and gastro-intestinal infections. It is also active as a bactericide on at least S^ pneumoniae, even those being resistant to antibiotics. COMMENTS S. pneumoniae and H^ influenzae are important causes of morbi¬ dity and mortality in all age groups. Respiratory tract infec- tions, e.g., meningitis, otitis, and sinusitis are caused by bacteria which enter via the nasopharynx. Colonization at that site may thus be an important determinant of disease (18). The finding that a specific alpha-lactalbumin derived from human as well as bovine milk inhibits attachment of both species opens the possibility to prevent colonization by specific interfe¬ rence of attachment using these structures. The bactericidal effect is hereby of importance as well. The importance of the antimicrobial molecules is shown by the protection against infections which is seen in breast-fed ba¬ bies. Breast-fed babies have a reduced frequency of diarrhoea, upper respiratory tract infections and acute otitis media (AOM). The bacterial species discussed in this application are the most frequent bacterial causes of AOM, viz. Haemophilus influenzae and Streptococcus pneumoniae. As evident from the data shown the alpha-lactalbumin obtained from the human or bovine milk inhibits the attachment of S. pneumoniae and H. influenzae to human respiratory tract epi- thelial cells in vitro. Table 3 Bacterial adhesion to oropharyngeal cells after incubation with active human milk, casein, and casein fractions obtained after ion-exchange chromatography on DEAE-Trisacryl Adhesion S. pneumoniae H. influenzae Sample Mean ( % ) Mean ( % ) Saline control 150 100) 200 (100) Human milk 25 (17) 70 (35) Casein 4 (3) 10 (5) Pool VI 14 (9) 22 (11) Pool K 3 (2) 17 (9) Pool L 150 (100) 178 (89) Table 4 Bacterial adhesion to oropharyngeal cells after incubation with human alpha-lactalbumin and the fractions obtained after ion- exchange chromatography and gel chromatography. Adhesion S. pneumoniae H. influenzae Sample Mean ( % ) Mean (%) Saline control 138 (100) 130 (100) Human alpha-lactalbumin 124 (90) 110 (85) Pool LA- 4 (3) 9 (7) Pool LA 128 (93) 76 (58) Table 5 Bacterial adhesion to oropharyngeal cells after incubation with bovine alpha-lactalbumin and the fractions obtained after ion- exchange chromatography and gel chromatography. Adhesion S. pneumoniae H. influenzae Sample Mean ( % ) Mean (%) Saline control 138 (100) 130 (100) Bovine alpha-lactalbumin 130 (94) 99 (76) Pool BL-, 3 (2) 18 (14) APPLICATIONS The alpha-lactalbumin of the present invention can be admini¬ stered in the form of an oral mucosal dosage unit, an inject- able composition, or a topical composition. In any case the protein is normally administered together with commonly known carriers, fillers and/or expedients, which are pharmaceutically acceptable . In case the protein is administered in the form of a solution for topical use the solution contains an emulsifying agent for the protein together with an diluent which can be sprayed into the nasopharynx, or can be inhaled in the form of a mist into the upper respiratory airways. In oral- use -the protein is normally administered together with a carrier, which may be a solid, semi-solid or liquid diluent or a capsule. These pharmaceutical preparations are a further object of the present invention. Usually the amount of active compound is between 0.1 to 99 % by weight of the preparation, preferably between 0.5 to 20 % by weight in preparations for injection and between 2 and 50 % by weight in preparations for oral administration. In pharmaceutical preparations containing a protein of the pre sent invention in the form of dosage units for oral admi¬ nistration the compound may be mixed with a solid, pulverulent carrier, as e.g. with lactose, saccharose, sorbitol, mannitol, starch, such as potatoe starch, corn starch, amylopectin, cel¬ lulose derivatives or gelatine, as well as with an antifrictio agent such as magnesium stearate, calcium stearate, polyethy¬ lene glycol waxes or the like, and be pressed into tablets. Multiple-unit-dosage granules can be prepared as well. Tablets and granules of the above cores can be coated with concentrate solutions of sugar, etc. The cores can also be coated with pol ymers which change the dissolution rate in the gastrointes¬ tinal tract, such as anionic -polymers having a pk of above cl 5.5. Such polymers are hydroxypropylmethyl cellulose phtalate, cellulose acetate phtalate, and polymers sold under the trade mark Eudragit S100 and L100. In the preparation of gelatine capsules these can ' be soft or hard. In the former case the active compound is mixed with an oil, and the latter case the multiple-unit-dosage granules are filled therein . Liquid preparations for oral administration can be present in the form of syrups or suspensions, e.g., solutions containing from about 0.2 % by weight to about 20 % by weight of the ac¬ tive compound disclosed, and glycerol and propylene glycol. If desired, such preparations can contain colouring agents, fla¬ vouring agents, saccharine, and carboxymethyl cellulose as a thickening agent . The daily dose of the active compound varies and is dependent on the type of administrative route, but as a general rule it is 1 to 100 g/dose of active compound at peroral administ¬ ration, and 2 to 200 mg/dose in topical administration. The number of applications per 24 hrs depend of the administration route, but may vary, e.g. in the case of a topical application in the nose from 3 to 8 times per 24 hrs, i.a., depending on the flow of phlegm produced by the body treated in therapeutic use. In prophylactic use the number may be on the lower side of the range given. The topical form can preferably be used in prophylactic treat¬ ment, preferably in connection with an infection caused by a rhinitis virus. The protein can also be used as an additive in infant food, particularly for prophylactic reasons, in order to supply the casein in an easy way to the child. Infants normally reject pharmaceuticals for different reasons. The food product can thus be in the form of a pulverulent porridge base, gruel base, milk substitute base, or more complex food product as of the Scotch collops type, comprising vegetables and meat pieces, of¬ ten in disintegrated form. In the case of protein administration to animals they are nor¬ mally added to the feedstuffs, which besides the protein con- tains commonly used nutrients. In accordance with a further aspect of the invention there is provided a process for determining the presence of S^ pneu o- cocci and H^ influenzae in a sample taken from the respiratory tract of an animal or human. This process is based on the tech¬ nique of determining the degree of interaction between the bac¬ teria of the sample and a composition of the present invention. Such interaction may be determined by inhibition or induction of the adherence of the bacteria to cells or other surfaces. REFERENCES 1. McKenzie, H.A., White, F.H. Jr Adv. Protein Chem. 41:173, 1991 2. Hopper, K.E. and McKenzie, H.A. Biochim. Biophys. Acta 295:352, 1973 3. Schmidt, D.V. and Ebner, K.E. Biochim. Biophys. Acta 263:714, 1972 4. Maynard, F. J. Dairy Res. 59:425, 1992 5. Barman, T.E. Eur. J. Biochim. 37:86, 1973 6. Readhead, K., Hill, T. and Mulloy , B. FEMS Microbiol Lett. 70:269, 1990 7. Gilin, F.D., Reiner, D.S. and Wang, C.S. Science 221:1290, 1983 8. Fiat, A.-M., and Jolles, P. Mol. Cell Biochem. 87:5, 1989 9. Matthews, T.H.J., Nair, C.D.G., Lawrence, M.K. and Tyrrell, D.A.J. Lancet, December, 25:1387, 1976 10. Andersson, B., Dahmen , J., Frejd, T., Leffler, H., Magnusson, G., Noori , G., and Svanborg, C, J. Exp. Med., 158:559, 1983 11. Svanborg, C, Aniansson, G., Mestecky, J., Sabharwal, H., • and Wold, A. In Immunology of milk and the neonate, J. Mestecky ed. Plenum Press, New York, 1991 12. Svanborg-Eden, C. and Svennerholm, A.-M., Infect. Immun. 22:790, 1978 13. in Microbial lectins and agglutinins, properties and bio¬ logical activity, Mirelman, D., Wiley, New York, 1986 14. Andersson, B., Porras, 0., Hansson, L.A., Lagergard, T. and Svanborg-Eden, C. J. Infect. Dis. 153:232, 1986 15. Aniansson, G., Andersson, B. , Lindstedt, R., and Svanborg, C, Microbial Pathogenesis 8 ^ , 365, 1990 16. Sabharwal, H., Hansson, C, Nilsson, A.K., Saraf, A., Lόnnerdahl, B. , and Svanborg, C. 1993, submitted 17. Lacks, S., and Hotchiss, R.D. Biochim. Biophys. Acta, 38:508, 1960 18. Branefors-Helander, P. Acta Pathol. Microbiol. Immunol. Scand. (B), 80:211, 1972 19. Porras, 0., Svanborg Eden, C, Lagergard, T., and Hansson, L. A. Eur. J. Clin. Microbiol., 4_, 310-15, 1985 20. Vanaman, T.C., Brew, K. , and Hill, R.L. J. Biol. Chem. 245:4583, 1970 21. Beachey , E.H. , J. Infect. Dis. 143, 325, 1981 22. Andersson, B. , Beachey, E.H., Tomasz, A., Tuomanen , E. , and Svanborg, C. , Microbial Pathogenesis, 4_, 267, 1988 23. Andersson, B. , Eriksson, B. , Falsen , E., et al Infect. Immun. 3_2 » 311-17, 1981

Claims: CLAIMS 1. The use of a multimeric alpha-lactalbumin in the preparation of therapeutically and/or prophylactically active antibacterial preparations, against infections, preferably of the respiratory tract, caused by bacteria, in particular S_^ pneumoniae and/or H. influenzae. 2. Use according to claim 1, wherein the multimeric alpha- lactalbumin is present in a mixture of monomeric, dimeric and trimeric forms . 3. Use according to claim 2, wherein the monomeric, dimeric, and trimeric forms are present in a weight ratio of about 15-7:5-2:1 4. Use according to claim 3, wherein the monomeric, dimeric, and trimeric forms are present in a weight ratio of about 10:3:1 5. Pharmaceutical composition comprising a therapeutically ac¬ tive amount of a protein as defined in any of claims 1-4 for the therapeutic and/or prophylactic treatment of infections, preferably in the respiratory tract, caused by bacteria, in particular S^_ pneumoniae and/or H^ influenzae. 6. Food and feed-stuff comprising an active amount of a protein as defined in any of claims 1-4 for the therapeutic and/or pro¬ phylactic treatment of infections, preferably in the respira¬ tory tract, caused by bacteria, in particular S^ pneumoniae and/or H^ influenzae. 7. Method for prophylactic and/or therapeutic treatment of in¬ fections caused by bacteria, in particular S^ pneumoniae and/or H. influenzae, wherein a therapeutically active amount of a protein as defined in any of claims 1-4 is administered to mam¬ mals, including humans, optionally in combination with the¬ rapeutically inert expedients or nutrients. 8. Method according to claim 7, wherein the alpha-lactalbumin is administered to prevent adhesion of virulent bacteria. 9. Method according to claim 7, wherein the alpha-lactalbumin is administered to exert a bactericidal effect on the virulent bacteria. 10. Method according to claim 7, wherein the alpha-lactalbumin is administered to provide a bactericidal effect on a virulent bacteria. 11. Method for diagnosing infections caused by bacteria, in particular S^ pneumoniae and/or H^ influenzae, wherein a sample from the infected mammals, including man is extracted and de- termined with regard to adhesion visavi a protein as defined in any of claims 1-4. 12. Method for preparing a protein according to any of claims 1-4, wherein a monomeric alpha-lactalbumin is subjected to an ion-exchange chromatography. 13. Method according to claim 12, wherein the ion-exchange chromatography is carried out on a DEAE-Tris-acryl gel. 14. Method according to claim 13, wherein the eluting agent is NaCl having a linear gradient. 15. Method for preparing a therapeutically and/or prophylac¬ tically ■ active antibacterial preparation against infections caused, preferably in the respiratory tract by S^ pneumoniae and/or H. influenzae , whereby a therapeutically active amount of multimeric alpha-lactalbumin is combined with inert expe¬ dients .

Inventors: SABHARWAL HEMANT, SVANBORG CATHARINA, SABHARWAL, HEMANT, SVANBORG, CATHARINA
Applicants: SABHARWAL HEMANT, SVANBORG CATHARINA, SABHARWAL, HEMANT, SVANBORG, CATHARINA
Application year: 1994
Main classification: A61K38
IPC codes: A23J1, A23J3, A23K1, A23L1, A23L3, A61K35, A61K38, A61P11, A61P31, C07K1, C07K14, G01N33